A specific circuit in the midbrain detects stress and induces restorative sleep

In mice, social defeat stress (SDS), an ethological model for psychosocial stress, induces sleep. Such sleep could enable resilience, but how stress promotes sleep is unclear. Activity-dependent tagging revealed a subset of ventral tegmental area GABA-somatostatin (VTAVgat-Sst) cells that sense stress and drive NREM and REM sleep via the lateral hypothalamus, and also inhibit corticotropin-releasing factor (CRF) release in the paraventricular hypothalamus. Transient stress enhances the activity of VTAVgat-Sst cells for several hours, allowing them to exert their sleep effects persistently. Lesioning of VTAVgat-Sst cells abolished SDS-induced sleep; without it, anxiety and corticosterone levels remained elevated after stress. Thus, a specific circuit allows animals to restore mental and body functions via sleeping, potentially providing a refined route for treating anxiety disorders.

After injections, mice that were to undergo the sleep experiments were implanted with three gold-plated miniature screw electrodes (-1.5 mm Bregma, +1.5 mm midline; +1.5 mm Bregma, -1.5 mm midline; -1 mm Lambda, 0 mm midline -reference electrode) with two EMG wire (AS634, Cooner Wire, CA). The EMG electrodes were inserted between the neck musculatures. EEG-EMG connector for the Neurologger 2A was affixed to the skull with Orthodontic Resin power and Orthodontic Resin liquid (Tocdental, UK).
For the telemetry EEG and EMG surgery, the mice were anesthetized with isoflurane as above and implanted in the abdominal subcutaneous with wireless TL11M2-F20-EET device (Data Sciences International) biotelemetry transmitters. Four of the wires were attached subcutaneously to the neck of the mice through a guide cannula. Then, mice were placed into a stereotaxic instrument in a prone position. Next, two surface EEG electrodes (biopotential leads tethered to miniature stainless-steel screws) were implanted over the left frontal lobe (AP 0.2 mm, ML 1.5 mm, DV −0.1 mm) and the contralateral parietal lobe (AP −1.7 mm, ML −0.2 mm, DV −0.1 mm) and lowered until they contacted the dura of the skull, with dental cement to secure in place. Two biopotential EMG electrodes were embedded through the small incision, which was made at the oblique cervical muscle with a 21G needle and fixed with insoluble silk sutures. The incision site was sutured and treated with local anesthetic (2% lidocaine) and topical antibiotic. For EEG/EMG recording, mice were individually housed following surgery in standard Plexiglas home cages placed on RPC-1 PhysioTel receivers, which receive signals from the transmitters. RPC-1 PhysioTel receivers are concatenated to a data exchange matrix, which transmits continuous and synchronized EEG, EMG and motor activity.

Cannula Implantation and administration of orexin receptor antagonists
Mice were placed in an induction chamber with 1.2-1.4% isoflurane (Baxter Healthcare, Puerto Rico) vaporized by oxygen flowing at 1.0 L/min and then transferred and fixed to a stereotaxic frame while keeping the mice anesthetized by 0.8% isoflurane via a mask. After cleaning and and exposure of the skull, AAV-DIO-taCasp3-TEV was injected into the VTA of Sst-IRES-Cre mice as described above. Three weeks after virus injection, a guide cannula (Stoelting, Co., US) was stereotaxically directed into the lateral cerebral ventricle (ML = −1.00 mm, AP = 0.50 mm, DV = −2.00 mm). The mice were allowed to recover for at least 7 days after the surgery.

Behavioral protocols
Social defeat stress, the non-stressful procedure, restraint stress, exposure to novel environments, voluntary wheel running, forced treadmill running and activity-tagging all took place one hour before the start of "lights on". The cage-change mild stress procedure was conducted at the beginning of "lights on". Mice were habituated for 10 min in the experimental room for a continuous period of 3 days before the experiments. Open-field tests (OFT) and elevated plus-maze (EPM) were performed at specific time points below: 1, before control or SDS (1-h before the start of "lights on"); 2, straight after control or SDS (at the beginning of "lights on"); 3, after control or SDS followed by 4-h home cage sleep or 4-h sleep deprivation (4-h after "lights on").
Social defeat stress was performed as reported with small modifications (Fig. S1A) (50).
Before performing social defeat experiments, male CD-1 retired breeder mice at 4-6 months of age, were singly housed, allowing habituation for a minimum of 7 d prior to screening. The appropriate CD-1 aggressive mice were selected from the 3-d screening process to meet the social defeat criterion (51). For screening the aggressors, C57BL/6j mice between 8 and 20 weeks of age were used. The CD-1 mice that showed extreme aggressive behavior or no aggression were excluded from experiments. Only mice showing aggression within 1 min and persistent aggressive intention were used in social defeat sessions (51).
For social defeat stress, an intruder mouse (experimental mouse) was introduced into the home cage of the CD1 resident mouse for 5 min. During these 5 min, there was usually 5-8 conflicts.
Then a transparent partition was used to separate the intruder and resident mice, but the intruder remained in olfactory, visual and auditory contact with the resident for 10 min. During the social defeat sessions, the procedure was repeated four times at 15 min intervals, for a total of 60 min (Fig. S1A). At the end of the stress procedure, the intruder exhibited an apparent freezing behavior or a submissive posture. Normally, no wound was found on intruders following this stress procedure; however, when the intruder mouse had visible wounds, the intruder and corresponding CD-1 mouse were excluded. For the controls, the intruder mouse was placed into the home cage of the aggressive resident mouse without contact for 60 min using a transparent partition to separate the intruder and resident mice, but remained in olfactory, visual and auditory contact with the resident (Fig. S1A). Video recording was performed with recordings of fiber photometry signals.
Non-stressful procedure. This protocol was used as the control for the physical movements during the social defeat stress. In detail, an intruder mouse (experimental mouse) was introduced into the home cage of the juvenile (younger) mouse for 5 min. During these 5 min, the mice experienced movements, physical activity and social interaction but typically had no conflicts. If the mice had any conflicts, the intruder and corresponding juvenile mouse were excluded. Then a transparent partition was used to separate the intruder and resident mice, but the intruder remained in olfactory, visual and auditory contact with the resident for 10 min.
During the non-stressful sessions, the procedure was repeated four times at 15 min intervals, for a total of 60 min (Fig. S1B). At the end of the non-stressful procedure, the intruders exhibited normal behaviors without having freezing behavior or a submissive posture. For the controls, the intruder mouse was placed into the home cage of the juvenile resident mouse without contact for 60 min using a transparent partition to separate the intruder and resident mice, but remained in olfactory, visual and auditory contact with the resident (Fig. S1B).
Restraint stress. The restraint paradigm was performed as previously described (7). Mice were individually placed into a well-ventilated 50-ml Falcon conical tube with a narrow open window on top to allow for movement and sliding of the fiber patch cord. During the entire restraint procedure, mice were in a natural body position without physical harm. For the control experiment of long-term fiber photometry, mice were placed into a 5L glass cylinder for 60 min without any restraint.
Cage-change mild stress produce was conducted as reported (52). At the beginning of ''lights on'', animals' home cages were exchanged with new cages with fresh bedding and food in the holding room. For the control experiment of long-term fiber photometry, mice were in the home cages for 60 min.
Novel environments. Mice were placed in a novel cage with a novel object in the experimental room for 60 min (Fig. S1C). For the controls, the mice were in their home cages in the holding room (Fig. S1C).
Novel objects. An object was placed in the resident mouse's home cage in the experimental room. Video recording was performed with recordings of fiber photometry signals.
Mild sleep deprivation was performed as we previously reported (39). Mice were sleep deprived at the start of "lights on" for 4-h using novel cages and novel objects. When mice showed signs of drowsiness, a soft brush was used to touch the mouse for 1-2 s. Forced treadmill running. The treadmill exercise regimen was adapted from previous study (53). To reduce the amount of stress to the animals, prior to the running experiment, mice were acclimatized to the stationary treadmill apparatus (Shanghai Yuyan Instruments Co., Ltd.) for 10 min, increasing to a speed of 4-10 m/min on three consecutive days. After this adaption period, mice experienced exercise training for 60 min, with the treadmill set at a speed of 6 m/min. The protocol corresponded to approximately 50% of maximal oxygen consumption.

Open-field tests
The Open Field Test (OFT) was used to test the anxiety-like behaviors of mice as reported (54).

Elevated plus-maze
The Elevated Plus-Maze test (EPM) was used to test the anxiety-like behaviors of the mice, as we previously performed (55). Following the Open Field Test, the experimental mice were

Activity-tagging
The activity-tagging procedure was performed as we previously reported (39,40,56). Vgat-IRES-Cre or Sst-IRES-Cre mice were placed on doxycycline (200 mg/kg) (Envigo TD.09265) for at least one week prior to the virus injection. Mice were injected with the activity-tagging transgenes. 2-4 weeks after injection, mice were taken off doxycycline 24-h before activitytagging (mice were kept undisturbed during this period). VTA Vgat or VTA Sst neurons were allowed to become tagged while mice experienced 1-h social defeat (SDS), or the non-stressful procedure, or voluntary wheel running, or forced treadmill running, or restraint stress, or cagechange or novel environments (novelty). For the control tagging, mice experienced 1-h control for social defeat or non-stressful procedure (using a transparent partition to separate the intruder and resident mice) (Behavioral protocols -social defeat stress and non-stressful procedure). To shut down the activity-tagging system, mice were put back on doxycycline for 5 to 7 days (without any disturbance for the first 2 days) before anatomical or behavioral assessments.

Assessment of the activity-tagging system
We assessed the efficacy and specificity of the activity-tagging by delivering transgenes into the VTA (Fig. S32A) or intersectionally into the VTA → LH pathway (Fig. S32B).
The efficacy of Dox: activity-tagging AAVs were injected into the VTA (Fig. S32A) or VTA → LH pathway (Fig. S32B) of Vgat-IRES-Cre mice. After injection, mice were constantly on Dox while tagged by SDS (On Dox → On Dox → SDS-tagging → On Dox). We did not observe many tagged cells, suggesting Dox is sufficient to inhibit the expression of the transgenes.
The specificity of tagging for SDS: activity-tagging AAVs were injected into the VTA (Fig.   S32A) or VTA → LH pathway (Fig. S32B) of Vgat-IRES-Cre mice. 24-h before activitytagging, Dox was removed, and mice underwent SDS, control or home cage procedure. After activity-tagging, mice were put back on dox for 5 to 7 days (On Dox → Off Dox → SDS-, control-or home cage-tagging → On Dox). Compared to SDS-tagging, we did not observe many control-or home cage-tagged cells, suggesting the cells that have been labeled in the experiments were specific for SDS.

Fiber photometry
Fiber photometry was performed as described (42,57). In detail, a Grass SD9 stimulator was used to control a 473-nm Diode-pumped solid-state (DPSS) blue laser with a fiber coupler The photometry signals during the stress experiences were matched with video recordings. For each experiment, the photometry signal F was converted to DF/F by DF/F(t)=(F(t)-median (F))/median (F) (42). Mice with an implanted optical fiber were connected to a patch cord for recording of the Ca 2+ signal. After connection, the mice were habituated in their home cages, novel cages or other experimental cages for 30 min. In some recordings, we observed decay of the photometry signal at the beginning of the recordings. All the trials were conducted after the photometry signal became stable. For photometry recordings across brain states (vigilance states), the photometry signals were matched with EEG/EMG signals. The photometry signals were not due to movement, as the GFP signal originating from control AAV-DIO-GFP expression in VTA Vgat neurons did not change during the stress (Fig. S33).

Chemogenetic protocols
We used the clozapine-N-oxide (CNO) chemogenetic method (58). CNO (C0832, Sigma-Aldrich, dissolved in saline, 1 mg/kg) or saline was injected i.p. Mice were split into random groups that received either saline or CNO injection. To test the sleep-promoting effects, we injected saline or CNO during the "lights off" period when the mice were in the active phase.
In detail, for the activity-tagged VTA Vgat neurons or activity-tagged VTA Sst neurons with hM3Dq-mCherry, saline or CNO was given during the "lights off" period. For non-tagged VTA Sst neurons expressing hM3Dq-mCherry, saline or CNO was given during the "lights off" period.
To chemogenetically reactivate activity-tagged LH-projecting VTA Vgat neurons or VTA Sst neurons, saline or CNO was given during the "lights off" period. For the activity-tagged VTA Vgat neurons with hM4Di-mCherry, saline or CNO was given 30 min before stress producers. Mice experienced SDS 1-h before the "lights on" period. Therefore, saline or CNO was given to these mice 90 min before the "lights on" period.

Optogenetic protocols
We used ChR2-based optogenetics (59). For optogenetic experiments, after the fiber patch cord was connected to the laser generator, the optical power meter (SANWA Electric Instrument, Tokyo, Japan) was used to measure laser intensity, to which the optic fiber was attached to obtain laser intensity at 10-15 mW. Then, the optic fibers on the head of mice were concatenated to the fiber patch via the rotary joint (ThinkerTech, Nanjing, China). During the experiments, the mice were stimulated optically (ChR2, 473 nm, 20 Hz, 30ms duration) every 60 s with 30 s interval. To test the sleep-promoting effects, opto-stimulation was conducted when the mice were awake.

Sleep-wake behavior and EEG analysis
Mice were attached with mock Neurologgers before experiments. EEG and EMG signals were recorded using Neurologger 2A devices (60). Wake, NREM or REM sleep was automatically classified at 5-s epoch using sleep analysis software Spike2 (Cambridge Electronic Design) and then manually re-scored. Episode duration was analyzed using MATLAB (R2020b) as we previously described (42).
For telemetry EEG and EMG, EEG data were analyzed using the NeuroScore Software (DSI, Harvard Bioscience, Inc). Vigilance states, including NREM sleep, REM sleep, and wake states, were first automatically assigned to each 5-s epoch using an automated scoring algorithm and then manually scored. The duration and bouts of vigilance state were calculated.
Meanwhile, spectral power was quantified from the raw EEG data through a multitaper method for each vigilance state. Analysis was performed using a custom MATLAB script via the Chronux toolbox (http://chronux.org) (61).

Enzyme immunoassay of serum corticosterone
Mice were decapitated immediately at each time point after the non-stressful procedure, SDS, restraint stress or novel environments, and their control protocols, respectively. Blood was rapidly collected in 1.5-ml plastic tubes placed in ice and centrifuged at 1,500 × g for 20 min, and serum was collected and stored at −80°C until use. Serum samples were tested for corticosterone concentration using the Enzo ELISA kit (ADI-900-097; Enzo Life Sciences, Exeter, Devon, UK) according to the manufacturer's instructions.
Injector cannula were connected with micro-syringe with Polyethylene pipe filled with mineral oil. 500 nL of SB-334867 or TCS-OX2-29 was infused into the lateral cerebral ventricle through the injector cannula, respectively. Mice were allowed to move freely in their home cage during infusions. Afterwards, cannulas were left in place for an additional 2-3 min to allow drug diffusion. 20 min after drug infusion, SDS or behavioral tests were conducted.

Single-cell RT-qPCR from acute brain slice of the VTA
Single-cell RT-qPCR was performed as we previously described (42,56). SDS-tagged VTA Vgat or VTA Sst mice were killed by cervical dislocation. The brains were quickly removed and The Mann-Whitney U-test was used for non-parametric tests. All data are given as mean ± SEM. Statistical tests were run in "Origin 2021" (Origin Lab).  Unpaired t-test, **p < 0.01, ***p < 0.0001.          DG, dentate gyrus of the hippocampus; 4V, 4 th ventricle of the brain. Scale bar, 100 µm.
(D) The activity-tagging protocol for opto-reactivation of the tagged VTA Vgat → LH pathway.
(E) Opto-reactivation of SDS-tagged VTA Vgat → LH pathway from waking. EMG, EEG, spectra, percentage and time of wakefulness, NREM or REM sleep (n = 6 control-tagged mice; n = 9 SDS-tagged mice). Mann-Whitney test, **p < 0.01. Fig. 3) The protocol using rabies virus-based retrograde tracing for identification of presynaptic inputs to VTA Vgat neurons and a schematic color matrix showing the percentage of total inputs from the whole brain to the VTA Vgat neurons as determined by N2CDG rabies retrograde tracing.

Fig. S11 Presynaptic inputs to VTA Vgat neurons (Related to
The heat code indicates the percentage of total inputs as determined by counting the numbers of rabies-labeled presynaptic cells.           Unpaired t-test, ***p < 0.001.          Unpaired t-test, p > 0.05, n.s, not significant.  Paired t-test, p > 0.05.